Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
The tripeptide thiol glutathione (L-.gamma.-glutamyl-L-cysteinylglycine; GSH) is found within virtually all cells. It functions in metabolism, transport, and cellular protection. Specifically, for example, glutathione participates in transhydrogenation reactions that are involved in the formation and maintenance of the sulfhydryl group of other molecules (e.g., coenzyme A, various enzymes, and other proteins). Glutathione provides reducing capacity for various reactions, e.g., the formation of deoxyribonucleotides by ribonucleotide reductase. Glutathione also functions in the detoxification of hydrogen peroxide, other peroxides, and free radicals. In addition, glutathione plays a role in detoxification of a variety of foreign compounds which interact with glutathione and which are ultimately excreted in the form of mercapturic acids. Analogous derivatives of glutathione are formed with endogenous metabolites, e.g., in the metabolism of leukotrienes, prostaglandins, steroids, and melanins. There is also evidence that the .gamma.-glutamyl moiety of glutathione functions in the transport of amino acids (especially cysteine and certain neutral amino acids) and possibly also of peptides and amines.
Glutathione synthesis takes place within almost all animal cells and in those of many plants and microorganisms. Two enzymes required for the synthesis of this tripeptide from L-glutamate (.gamma.-glutamylcysteine synthetase and glutathione synthetase) have been isolated from a number of different sources (Dolphin et al. 1989; Snoke and Bloch 1952; Snoke 1955; Meister 1974).
Gamma-glutamylcysteine (.gamma.-glu-cys) synthetase catalyzes the rate-limiting step of GSH synthesis: ##STR1## Gamma-glutamylcysteine is feedback inhibited by GSH (Richman and Meister 1975; Huang et al. 1993).
Glutathione synthetase catalyzes the synthesis of GSH from .gamma.-glutamylcysteine and glycine: ##STR2## 5-Oxo-L-prolinase(5-OPase) catalyzes the ATP-dependent cleavage of 5-oxoproline to L-glutamate: ##STR3##
Glutathione synthetase deficiency in humans is associated with potentially serious health complications. Two general types of such deficiency have been observed (Meister and Larsson 1995). In one, an unstable form of GSH synthetase is expressed, leading to an apparently selective deficiency of GSH in the erythrocyte. In 5-oxoprolinuria, the result of another type of GSH synthetase deficiency, dramatic and potentially fatal metabolic consequences occur as a result of over-production of 5-oxoproline which leads to severe metabolic acidosis. In this condition, there is over-production of .gamma.-glutamylcysteine, whose synthesis is not feedback inhibited because of the low levels of GSH and possibly because there is induction of .gamma.-glutamylcysteine synthetase. .gamma.-Glutamylcysteine is converted by the action of .gamma.-glutamylcyclotransferase to cysteine and 5-oxoproline. Cysteine is used by .gamma.-glutamylcysteine synthetase (in a futile cycle), and 5-oxoproline accumulates in amounts that exceed the capacity of 5-oxoprolinase to convert it to glutamate. This leads to substantial accumulation of 5-oxoproline and to its urinary excretion in amounts that may be as high as 30 grams per day (normally &lt;0.14 g. per day) (Meister and Larsson 1995). Severe damage to the central nervous system, and even potentially death, are among the potential complications. There are also recent reports of another form of 5-oxoprolinuria, associated with a deficiency of 5-oxoprolinase.
Modifications of glutathione metabolism are sometimes desirable even in persons having normal glutathione levels. Such modifications may be achieved by administration of selective enzyme inhibitors to decrease intracellular glutathione levels, or by providing compounds that increase glutathione synthesis. Such effects are potentially useful, respectively, in chemotherapy and radiation therapy, and in protecting cells against the toxic effects of drugs, other foreign compounds and oxygen.
Modification of GSH metabolism to deplete or increase cellular GSH may serve various purposes. For instance, it has long been known that thiols protect cells against the effects of irradiation. Since decreasing cellular GSH makes cells more susceptible to irradiation, glutathione depletion is useful in chemotherapeutic situations in which the cells to be killed and the cells to be spared have substantially different quantitative requirements for GSH. Depletion of GSH by inhibition of its synthesis also serves as a valuable adjuvant in chemotherapy with drugs that are detoxified by reactions involving GSH.
Conversely, development of resistance to a drug or to radiation may be associated with an increase in cellular GSH. GSH serves effectively in the detoxification of many drugs, and it is known that a significant pathway of acetaminophen detoxification involves conjugation with GSH.
Treatment with a thiazolidine such as L-2-oxothiazolidine-4-carboxylic acid, may be of value to patients with liver disease and to premature infants who may be deficient in the utilization of methionine sulfur for cysteine formation, and thus in GSH synthesis. The effectiveness of such a thiazolidine as an intracellular cysteine precursor depends on the presence of 5-oxoprolinase, an enzyme activity found in almost all animal cells.
The cleavage of 5-oxoproline by 5-OPase to glutamate is highly unusual in that hydrolysis of ATP is required for the cleavage of a specific peptide bond (Meister et al. 1985). 5-OPase has been found in mammalian tissues (Van Der Werf et al. 1971), plants (Mazelis and Creveling 1978), and microorganisms (Van Der Werf and Meister 1974; Mooz and Wigglesworth 1976). Apparently homogeneous preparations of 5-OPase from rat kidney (Williamson and Meister 1982) and Pseudomonas putida (Seddon et al. 1984) have been obtained and were used for physical characterization and for studies of catalytic mechanism. 5-OPase from rat kidney is composed of two apparently identical subunits which exhibit a molecular mass of 142,000 Da on SDS-polyacrylamide gel electrophoresis (Williamson and Meister 1982). The enzyme is evidently a "sulfhydryl enzyme" (Van Der Werf et al. 1975) and has a number of sulfhydryl groups/monomer (Williamson and Meister 1982). The relationship between the essential sulfhydryl groups of the enzyme and its various catalytic activities has been probed (Williamson and Meister 1982). Unlike rat kidney 5-OPase, Pseudomonas putida 5-OPase is composed of two different, reversibly dissociable protein components, A and B (Seddon et al. 1984). Component A catalyzes an initial step in the reaction that involves 5-oxoproline and ATP (Seddon and Meister 1986). Component B may function as a catalyst that converts a phosphorylated form of 5-oxoproline to glutamate, or it might alter the conformation of Component A so as to facilitate the reaction (Li et al. 1988; Li et al. 1989).
Data are lacking, however, on the amino acid sequence of the 5-oxoprolinase enzyme from any mammalian source. Knowledge of the amino acid sequence of the enzyme and the cloning of the encoding cDNA are essential for further studies on the structure, mechanism of action, and physiological function of the enzyme.
A need continues to exist for the determination of the nucleotide and amino acid sequences of mammalian 5-oxoprolinase.